The long-term objective of this project is to develop a molecular-level understanding of the tissue selectivity of ligands that modulate the estrogen receptors (ERalpha and ERbeta). Drugs that target estrogen signaling pathways such as tamoxifen and raloxifene display antiestrogenic effects in certain tissues ans estrogenic effects in other tissues. Understanding the molecular basis of this tissue selectivity will be useful for designing safer and more effective-based therapeutics. The proposed experiments are designed to test the hypothesis that ER ligand pharmacology derives from the interplay of three parameters: ligand structure, receptor subtype (ERalpha and ERbeta), and the type of response element in the promoter of a given gene that is the site of regultion by the ER. The ER/AP-1 site is a response element where differential ligand activation profiles are observed for ERalpha and ERbeta. The specific aim is to map the regions of ERalpha and ERbeta that mediate this differential ligand activation. The second specific aim of the project is to probe the structural differences between raloxifene and tamoxifen to identify the chemical substructures of these drugs that correlate with the differences observed in ligand activation at an R/Ap-1 site. The ER/AP-1 assay that facilitated discovery of the differential ligand activation properties of ERalpha and ERbeta is an artificial cellular transcription assay that uses a test promoter driving transcription of a reporter gene. There is great interest now to ask whether endogenous in target tissues can be regulated in a similar way; i.e. in an ERbeta expressing tissue which genes are up-regulated by antiestrogens such as tamoxifen and raloxifene and down-regulated by estradiol? This question is addressed in specific aim 3 through experiments using differential gene expression array technology. The endogenous estrogen-regulated genes that are discovered with this approach will be characterized through experiments described in specific aim 4. In particular, the promoters of these genes will be analyzed to identify the type of response element that facilitates regulation through the ER.